The use of double-stranded RNA (dsRNA) to trigger RNA interference and thereby silence matching disease genes is being pursued as a therapeutic approach for many human diseases. However, it is difficult to deliver these RNA-based drugs into human cells. In contrast, when dsRNA is introduced into one cell in plants and in some animals such as the worm C. elegans, genes in distant cells are also silenced due to the transport between cells of forms of dsRNA called mobile RNA. This direct transport of RNA between cells represents a novel form of intercellular signaling that was accidentally discovered as part of gene silencing studies in the worm C. elegans and in plants. How RNA that is transcribed in the nucleus is secreted from animal cells to silence a target gene in distant cells s not well understood. Studies in C. elegans identified conserved proteins required for the uptake of mobile RNAs suggesting that similar mechanisms may exist in other animals, including mammals. Intriguingly, populations of extracellular RNAs have been found in human blood and are being pursued as diagnostic tools to assess the disease state of internal organs, which are presumably the source of such RNAs. The goals of this proposal are to use the tools available in C. elegans to uncover how dsRNA is modified to generate mobile RNAs that are secreted from cells and to analyze genes that control the export of such RNAs from cells. Using available mutants, we will take biochemical and genetic approaches to identify modified dsRNAs and the mobile RNAs that result from them. We will use new mutants we have isolated that are defective in the intercellular transport of RNAs to discover additional genes that control this process and analyze them. The completion of these aims can reveal how RNAs are modified for export from animal cells and can begin to assemble the pathway for intercellular signaling by the direct transport of RNA between animal cells. These studies are also relevant to understand the entirely unknown biology of extracellular RNAs, which can become powerful diagnostic tools for diseases of internal organs. Finally, if we understand what modifications to dsRNA and other cellular machinery enable the efficient entry of mobile RNAs into C. elegans cells, we can design similar modified dsRNAs for efficient delivery into human cells for RNA interference-based therapy.